Maintenance of physiological epidermal tissue turnover is supported by a small number of stem cells that self renew and generate differentiated progeny in what is generally thought to be a unidirectional differentiation program. However, our recent studies showing that differentiated keratinocytes can and do revert to self renewing stem cells challenges this long held view. Using lineage tracing studies of labeled involucrin expressing keratinocytes, we have shown that differentiated keratinocytes can be induced to form a multi-lineage, hair-bearing skin epithelia. These studies suggest that differentiated keratinocytes possess an inducible proliferative capacity that is activated in response to cues from the microenvironment. This reversibility could be recapitulated in primary cultures of mouse keratinocytes by switching growth-arrested, differentiated keratinocytes to low calcium conditions thereby inducing reversion to a basal cell phenotype and programming. Using this model we identified the protein kinase D1 (PKD1) signaling pathway as key to this de-differentiation. PKD is a novel, multifunctional, signaling enzyme complex that is involved in the regulation of many important cellular functions including cell survival, proliferation, migration, differentiation, membrane trafficking and protein transport. Moreover, PKD1 is implicated in several pathological processes including stress-dependent cardiac hypertrophy, pathological angiogenesis and tumor cell proliferation and metastasis. Surprisingly, despite expression of all three PKD isoforms in keratinocytes, little is known about the role of these enzymes in skin. Our preliminary data show that specific knockdown of PKD1 blocked dedifferentiation in differentiated keratinocytes while having no effect on cell proliferation in cultures of replicating keratinocytes. These data suggest unique and redundant roles for PKD isoforms. We intend to use the exploratory nature of this application to investigate the role of PKDs in skin epithelia and to establish the in vivo correlate of this phenomenon in intact skin. In the first aim we characterize the functional significance of PKD signaling in regulating skin epithelia structure and function. We will take advantage of the availability of the PKDloxp/loxp mouse to generate a conditional PKD1 knockout to examine the effects of PKD1 loss on skin epithelia structure and function. We then use PKD1-deficient keratinocytes as a platform to identify the unique and redundant functions of other PKD isoforms. In the second aim we will extend the use of conditional PKD1 knockout mice to determine the contribution of PKD1 to the hyperplastic/adaptive responses of epidermis. Defining a critical role for PKD enzymes in epidermal adaptive responses will provide an opportunity to develop novel therapeutic strategies to suppress uncontrolled growth, to promote tissue regeneration after injury and to maintain stem cell activity during aging. PUBLIC HEALTH RELEVANCE: De-differentiation, the progression of cells from a more to a less differentiated state, is one of the most important principles in tissue regeneration in lower vertebrates. In mammals, it may contribute to maintenance of the stem cell population or play a role in tissue regeneration following injury. Insights into de-differentiation could form the basis for novel strategies to suppress uncontrolled growth, to promote tissue regeneration after injury and to maintain stem cell activity during aging.